title: Heritable targeted inactivation of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene using zinc-finger nucleases.
abstract: Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.
authors.affiliation: INRA, UR1037, LPGP, Fish Physiology and Genomics, 35000, Rennes, France.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Yano
authors.forename: Barbara
authors.initials: B
authors.lastname: Nicol
authors.forename: Elodie
authors.initials: E
authors.lastname: Jouanno
authors.forename: Yann
authors.initials: Y
authors.lastname: Guiguen
mesh_terms: D000818:Animals; D001483:Base Sequence; D004720:Endonucleases / Q000235:genetics*; D005260:Female; D018390:Gene Targeting / Q000379:methods*; D024282:Genes, sry / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D008297:Male; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000196:embryology / Q000235:genetics*; D012727:Sex Characteristics; D019849:Sex Determination Processes / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000235:genetics*; D029961:Zebrafish Proteins / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D016212:Transforming Growth Factor beta; D029961:Zebrafish Proteins; C571097:gonadal soma-derived factor, zebrafish; D004720:Endonucleases